Platinum complexes and methods of use

ABSTRACT

The subject invention concerns platinum complexes that exhibit antitumor cell and/or antiparasitic activity. The subject invention also concerns the use of platinum complexes of the invention to treat oncological and inflammatory disorders. The platinum complexes of the invention can also be used to treat or prevent infection by a virus or a bacterial or parasitic organism in vivo or in vitro.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is a divisional of U.S. application Ser. No.10/918,769, filed Aug. 13, 2004, which claims the benefit of U.S.Provisional Application Ser. No. 60/481,226, filed Aug. 13, 2003, U.S.Provisional Application Ser. No. 60/515,580, filed Oct. 30, 2003, U.S.Provisional Application Ser. No. 60/525,295, filed Nov. 25, 2003, andU.S. Provisional Application Ser. No. 60/519,943, filed Nov. 14, 2003,the disclosure of each of which is incorporated herein by reference inits entirety.

BACKGROUND OF THE INVENTION

Cellular responses to growth factors and cytokines are characterized byactivation of the Signal Transducer and Activator of Transcription(STAT) family of cytoplasmic transcription factors (Darnell, 1997;Darnell et al., 1994; Schindler et al., 1995; Stark et al., 1998;Smithgall et al., 2000; Akira, 2000; Hirano et al., 2000; Bromberg etal., 1996; Fukada et al., 1996; Kotenko et al, 2000). STATs areactivated at a very early stage involving protein tyrosine kinasephosphorylation of tyrosine of growth factor receptors,receptor-associated Janus kinase (Jaks) or Src kinase families. This inturn induces phosphotyrosine (pTyr)-SH2 interactions between two STATmonomers in the formation of dimers, translocation to the nucleus, andbinding to specific DNA response elements regulating gene expressionessential for cell proliferation, differentiation, development andsurvival.

Normal STAT activation is tightly-regulated and has a short duration,which is in keeping with normal cellular requirements for mounting aresponse to external stimuli. However, persistent activation of specificSTAT proteins, particularly Stat3 and Stat5, occurs with high frequencyin some tumors and has a causal role in malignant transformation bypromoting growth and survival of transformed and tumor cells, includingthose breast, prostate and head and neck squamous carcinoma cells,lymphomas and leukemias (Bromberg et al., 1999; Turkson et al., 1998;Bromberg et al., 1998; Catlett-Falcone et al., 1999a; Garcia et al.,2001; Grandis et al., 2000; Grandis et al., 1998; Nielsen et al., 1997;Nielsen et al., 1999; Epling-Burnette et al., 2001; reviewed in Bowmanet al., 2000; Turkson et al., 2000; Song et al., 2000; Coffer et al.,2000; Lin et al., 2000; Catlett-Falcone et al., 1999b; Garcia et al.,1998). Of clinical importance, blockade of aberrant Stat3 signaling inmalignant cells and whole tumors that contain them induces apoptosis andtumor regression.

Platinum complexes, the prototype of cisplatin (Cis-Pt), have beenwidely used as active anticancer agents (Ardizzoni et al., 1999; Nitiss,2002) in a variety of human tumors, including testicular, ovarian,bladder carcinoma, head and neck, and non-small cell lung cancers. Theoutcome of treatments with cisplatin and other platinum-containingcompounds is strongly linked to their alkylating effects on DNA.However, the potential impact of platinum-complex-based therapy oncellular signaling and the therapeutic importance of such interactionshave yet to be explored. Reports show that cisplatin induces activationof members of the mitogen-activated protein kinase (MAPK) pathways(Persons et al., 1999; Sanchez-Perez et al., 1998), which may influencedrug-induced apoptosis.

BRIEF SUMMARY OF THE INVENTION

The subject invention concerns platinum complexes and uses thereof. Theplatinum complexes of the invention can be used to treat oncological,viral, bacterial, and parasitic disease conditions.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows the results in graph form from an MTT assay. Cisplatin isalso designated as “Cis-Pt.”

FIG. 2 shows the results in graph form from an MTT assay. Cisplatin isalso designated as “Cis-Pt.”

FIG. 3 shows the results in graph form from an XTT assay. Cisplatin isalso designated as “Cis-Pt.”

FIG. 4 shows the results in graph form from an MTT assay. Cisplatin isalso designated as “Cis-Pt.”

FIGS. 5A-C show inertness of CPA-7 platinum complex to reduction byglutathione. 20 μm CPA-7 in PBS/20% DMSO with 10 mm glutathione. Asshown in FIG. 5A, linear portions were measured at night (dark room)whereas reduction occurred in daytime. CPA-7 is not appreciably reducedby GSH but by light. As shown in FIG. 5B-C, scanning kinetics over twohours shows little change. Reduction is attributed to room light.

FIG. 6 shows the results in graph form from an MTT assay. Cisplatin isalso designated as “Cis-Pt.”

FIG. 7 shows the results in graph form from an XTT assay. Cisplatin isalso designated as “Cis-Pt.”

FIG. 8 shows the results in graph form from an MTT assay. The figurelegend identifies the Pt(NO₂)(NH₃)₂(Cl)₂A platinum complex by the “A”substituent identified herein. Cisplatin is also designated as “Cis-Pt.”Two different isolates of the platinum (IV) complex substituted withSafranin were isolated (referred to herein as Safranin 1 and Safranin 2)and tested in the assay.

FIG. 9 shows the results in graph form from an MTT assay. The figurelegend identifies the Pt(NO₂)(NH₃)₂(Cl)₂A platinum complex by the “A”substituent identified herein. Cisplatin is also designated as “Cis-Pt.”Two different isolates of the platinum (IV) complex substituted withSafranin were isolated (referred to herein as Safranin 1 and Safranin 2)and tested in the assay.

FIG. 10 shows the results in graph form from an XTT assay. The figurelegend identifies the Pt(NO₂)(NH₃)₂(Cl)₂A platinum complex by the “A”substituent identified herein.

FIG. 11 shows the results in graph form from an MTT assay. The figurelegend identifies the Pt(NO₂)(NH₃)₂(Cl)₂A platinum complex by the “A”substituent identified herein. Cisplatin is also designated as “Cis-Pt.”

FIGS. 12A-B are photographs showing nuclear extracts containingactivated Stat1 and Stat3 are treated with the indicated concentrationsof other platinum (IV) complexes (designated herein as HK 104, HK 105,HK 106, HK 107, HK 108, HK 109, HK 110, HK 111, and HK 112) for 30 minat room temperature prior to incubation with radiolabeled hSIEoligonucleotide probe. Stat1 and Stat3 binding activities to hSIE probeare shown.

BRIEF DESCRIPTION OF THE SEQUENCE

SEQ ID NO:1 is the nucleotide sequence of an oligonucleotide probe.

DETAILED DISCLOSURE OF THE INVENTION

The subject invention concerns platinum complexes and uses thereof.Platinum complexes of the invention can induce apoptosis and/or inhibittumor cell growth and can also be used to treat cancers. The platinumcomplexes of the invention also can be used as antiviral, antibacterial,and antiparasitic agents. It has been suggested that cellularcytotoxicity of platinum (IV) compounds is a result of platinum (IV)compounds being reduced to platinum (II) in the cell. Surprisingly,platinum (IV) complexes of the present invention may not require thistype of reduction in the cells to have a cytotoxic effect. Therefore,the platinum complexes of the present invention are distinct fromplatinum compounds in the art by maintaining their correct oxidativeconformation as platinum (IV) compounds which are more effective thanthe existing platinum (II) compounds. In addition, platinum complexes ofthe invention can also form nitric oxide in the cells as radicalsthereby killing the cells through the formation of oxide radicals.

Platinum complexes of the invention include those complexes having thestructure shown in formula I:

whereinX and Y are, independently, any halogen, —NO₂, —ONO, or the structure:

or X and Y together form the structure:

R¹ is —NO₂ or —ONO;R² is any halogen, —OH, —ONO, —ONO₂, —COR¹⁰, —OPO₃R¹⁰R¹¹, —OSO₃H,—OSeOOH, —SeOOH,—AsO₂, —OAsO₂, —NR¹⁰R¹¹, —NHR¹⁰R¹¹, —OOCR¹⁵, alkyl, alkoxy, cycloalkyl,cycloalkoxy, aryl, aryloxy, alkycarbonyl, alkoxycarbonyl,cycloalkylcarbonyl, heteroalkyl, heterocycloalkyl,heterocycloalkylcarbonyl, heteroaryl, arylcarbonyl, heteroarylcarbonyl,aryloxycarbonyl, heteroaryloxycarbonyl, heterocycloalkoxy, orheterocycloalkoxycarbonyl, any of which can be optionally substitutedwith any halogen, —COOH, —OH, —NO₂, —NH₂, —N-alkyl, alkyl, alkoxy,cycloalkyl, cycloalkoxy, aryl, aryloxy, alkycarbonyl, alkoxycarbonyl,cycloalkylcarbonyl, heteroalkyl, heterocycloalkyl,heterocycloalkylcarbonyl, heteroaryl, arylcarbonyl, heteroarylcarbonyl,aryloxycarbonyl, heteroaryloxycarbonyl, heterocycloalkoxy, orheterocycloalkoxycarbonyl, or the structure:

any of which can be substituted with any halogen, —NH₂, —COOH, —OH,alkoxy, cycloalkoxy;R³ is, independently, —NH₃, or —NHR⁷;R⁷ is H, C₁₋₆ alkyl, alkoxy, or aryl, optionally substituted with —NO₂or —COOH;R¹⁰ and R¹¹ are, independently, H, —NH₂, —OH, —NHR⁷, CONHR⁷, CON(R⁷)₂,C₁₋₆ alkyl, aryl, or heteroaryl, any of which can be optionallysubstituted with any halogen, —COOH, —OH, —NO₂,—NH₂, alkyl, alkoxy, cycloalkyl, cycloalkoxy, aryl, aryloxy,alkycarbonyl, alkoxycarbonyl, cycloalkylcarbonyl, heteroalkyl,heterocycloalkyl, heterocycloalkylcarbonyl, heteroaryl, arylcarbonyl,heteroarylcarbonyl, aryloxycarbonyl, heteroaryloxycarbonyl,heterocycloalkoxy, or heterocycloalkoxycarbonyl;R¹⁵ is alkyl, alkoxy, cycloalkyl, cycloalkoxy, aryl, aryloxy,alkycarbonyl, alkoxycarbonyl, cycloalkylcarbonyl, heteroalkyl,heterocycloalkyl, heterocycloalkylcarbonyl, heteroaryl, arylcarbonyl,heteroarylcarbonyl, aryloxycarbonyl, heteroaryloxycarbonyl,heterocycloalkoxy, or heterocycloalkoxycarbonyl, any of which can beoptionally substituted with any halogen, —COOH, —OH, —NO₂, —NH₂,—N-alkyl, alkyl, alkoxy, cycloalkyl, cycloalkoxy, aryl, aryloxy,alkycarbonyl, alkoxycarbonyl, cycloalkylcarbonyl, heteroalkyl,heterocycloalkyl, heterocycloalkylcarbonyl, heteroaryl, arylcarbonyl,heteroarylcarbonyl, aryloxycarbonyl, heteroaryloxycarbonyl,heterocycloalkoxy, or heterocycloalkoxycarbonyl;or a pharmaceutically acceptable salt thereof.

In one embodiment, X and Y can be, independently, fluorine (F), chlorine(Cl), bromine (Br) or iodine (I). In an exemplified embodiment, X is Cland Y is Cl.

In one embodiment, R¹ is —NO₂, R² is Cl and R³ is —NH₃.

Platinum complexes of the invention can also have the structure shown informula II:

whereinX and Y are, independently, any halogen, or the structure:

or X and Y together form the structure:

R⁴ is —NO₂ or —ONO;R⁵ is any halogen, —OH, —ONO, —ONO₂, —COR¹⁰, —OPO₃R¹⁰R¹¹, —OSO₃H,—OSeOOH, SeOOH,—AsO₂, —OAsO₂, —NR¹⁰R¹¹, —NHR¹⁰R¹¹, —OOCR¹⁵, alkyl, alkoxy, cycloalkyl,cycloalkoxy, aryl, aryloxy, alkycarbonyl, alkoxycarbonyl,cycloalkylcarbonyl, heteroalkyl, heterocycloalkyl,heterocycloalkylcarbonyl, heteroaryl, arylcarbonyl, heteroarylcarbonyl,aryloxycarbonyl, heteroaryloxycarbonyl, heterocycloalkoxy, orheterocycloalkoxycarbonyl, any of which can be optionally substitutedwith any halogen, —COOH, —OH, —NO₂, —NH₂, —N-alkyl, alkyl, alkoxy,cycloalkyl, cycloalkoxy, aryl, aryloxy, alkycarbonyl, alkoxycarbonyl,cycloalkylcarbonyl, heteroalkyl, heterocycloalkyl,heterocycloalkylcarbonyl, heteroaryl, arylcarbonyl, heteroarylcarbonyl,aryloxycarbonyl, heteroaryloxycarbonyl, heterocycloalkoxy, orheterocycloalkoxycarbonyl, or the structure:

any of which can be substituted with any halogen, —NH₂, —COOH, —OH, or Yand R⁵ form the structure:

or X, Y, and R⁵ together form the structure:

R⁶ is, independently, NH₂ or NH;R⁷ is H, C₁₋₆ alkyl, alkoxy, aryl, optionally substituted with —NO₂ or—COOH;R⁸ and R⁹ are, independently, H, C₁₋₆ alkyl, or —OH, any of which can beoptionally substituted with any halogen, —COOH, —OH, —NO₂, —NH₂, alkyl,alkoxy, cycloalkyl, cycloalkoxy, aryl, aryloxy, alkycarbonyl,alkoxycarbonyl, cycloalkylcarbonyl, heteroalkyl, heterocycloalkyl,heterocycloalkylcarbonyl, heteroaryl, arylcarbonyl, heteroarylcarbonyl,aryloxycarbonyl, heteroaryloxycarbonyl, heterocycloalkoxy, orheterocycloalkoxycarbonyl; R¹⁰ and R¹¹ are, independently, H, —NH₂, —OH,—NHR⁷, CONHR⁷, CON(R⁷)₂, C₁₋₆ alkyl, aryl, or heteroaryl, any of whichcan be optionally substituted with any halogen, —COOH, —OH, —NO₂,—NH₂, alkyl, alkoxy, cycloalkyl, cycloalkoxy, aryl, aryloxy,alkycarbonyl, alkoxycarbonyl, cycloalkylcarbonyl, heteroalkyl,heterocycloalkyl, heterocycloalkylcarbonyl, heteroaryl, arylcarbonyl,heteroarylcarbonyl, aryloxycarbonyl, heteroaryloxycarbonyl,heterocycloalkoxy, or heterocycloalkoxycarbonyl;R¹² and R¹³ are, independently, H or C₁₋₆ alkyl, or R¹² and R¹³ togetherform an aryl, cycloalkyl, heterocycloalkyl, or heteroaryl, any of whichcan be optionally substituted with any halogen, —COOH, —OH, —NO₂, —NH₂,alkyl, alkoxy, cycloalkyl, cycloalkoxy, aryl, aryloxy, alkycarbonyl,alkoxycarbonyl, cycloalkylcarbonyl, heteroalkyl, heterocycloalkyl,heterocycloalkylcarbonyl, heteroaryl, arylcarbonyl, heteroarylcarbonyl,aryloxycarbonyl, heteroaryloxycarbonyl, heterocycloalkoxy, orheterocycloalkoxycarbonyl;R¹⁵ is alkyl, alkoxy, cycloalkyl, cycloalkoxy, aryl, aryloxy,alkycarbonyl, alkoxycarbonyl, cycloalkylcarbonyl, heteroalkyl,heterocycloalkyl, heterocycloalkylcarbonyl, heteroaryl, arylcarbonyl,heteroarylcarbonyl, aryloxycarbonyl, heteroaryloxycarbonyl,heterocycloalkoxy, or heterocycloalkoxycarbonyl, any of which can beoptionally substituted with any halogen, —COOH, —OH, —NO₂, —NH₂,—N-alkyl, alkyl, alkoxy, cycloalkyl, cycloalkoxy, aryl, aryloxy,alkycarbonyl, alkoxycarbonyl, cycloalkylcarbonyl, heteroalkyl,heterocycloalkyl, heterocycloalkylcarbonyl, heteroaryl, arylcarbonyl,heteroarylcarbonyl, aryloxycarbonyl, heteroaryloxycarbonyl,heterocycloalkoxy, or heterocycloalkoxycarbonyl;n is any integer from 0 to 6;or a pharmaceutically acceptable salt thereof.

In one embodiment, X and Y can be, independently, fluorine (F), chlorine(Cl), bromine (Br) or iodine (I). In an exemplified embodiment, X is Cland Y is Cl.

In one embodiment, R⁴ is —NO₂, R⁵ is Cl, R⁶ is —NH₂, and n is 0.

Platinum complexes of the invention can also have the structure shown informula III or IV:

whereinX and Y are, independently, any halogen, —NO₂, —ONO, or X and Y togetherform the structure:

R⁶ is, independently, NO₂, N, NH, or NH₂;R⁸ and R⁹ are, independently, H, C₁₋₆ alkyl, or —OH, any of which can beoptionally substituted with any halogen, —COOH, —OH, —NO₂, —NH₂, alkyl,alkoxy, cycloalkyl, cycloalkoxy, aryl, aryloxy, alkycarbonyl,alkoxycarbonyl, cycloalkylcarbonyl, heteroalkyl, heterocycloalkyl,heterocycloalkylcarbonyl, heteroaryl, arylcarbonyl, heteroarylcarbonyl,aryloxycarbonyl, heteroaryloxycarbonyl, heterocycloalkoxy, orheterocycloalkoxycarbonyl;R¹² and R¹³ are, independently, H or C₁₋₆ alkyl, or R¹² and R¹³ togetherform an aryl, cycloalkyl, heterocycloalkyl, or heteroaryl, any of whichcan be optionally substituted with any halogen, —COOH, —OH, —NO₂, —NH₂,alkyl, alkoxy, cycloalkyl, cycloalkoxy, aryl, aryloxy, alkycarbonyl,alkoxycarbonyl, cycloalkylcarbonyl, heteroalkyl, heterocycloalkyl,heterocycloalkylcarbonyl, heteroaryl, arylcarbonyl, heteroarylcarbonyl,aryloxycarbonyl, heteroaryloxycarbonyl, heterocycloalkoxy, orheterocycloalkoxycarbonyl;n is any integer from 0 to 6;or a pharmaceutically acceptable salt thereof.

In one embodiment, X and Y can be, independently, fluorine (F), chlorine(Cl), bromine (Br) or iodine (I). In an exemplified embodiment, X is Cland Y is Cl.

Also contemplated within the scope of the invention are platinumcomplexes that are not defined by formula I or formula II but that arespecifically exemplified in the Table 5 presented herein. Exemplifiedembodiments of platinum complexes of the invention are shown in Table 5.The chemical structure of a complex along with a designation name (e.g.,CPA-XX) is shown in the Table. Alternative designation names (e.g.,HKXXX) of a complex are shown in parentheses.

Platinum complexes of the invention also include those complexes havingthe structure shown in formula V or formula VI:

whereinX and Y are, independently, any halogen, —OH, H₂O, or —SO(CH₃)₂; and Acan be any of the following:

and whereinR¹ is, independently, NH₂ or NH;R² and R³ are, independently, H, —OH, C₁₋₆ alkyl, alkoxy, cycloalkyl,aryloxy, cycloalkoxy, aryl, heteroalkyl, heterocycloalkyl, heteroaryl,arylcarbonyl, and heteroarylcarbonyl, any of which can be optionallysubstituted with alkyl, alkoxy, cycloalkyl, aryloxy, cycloalkoxy, aryl,heteroalkyl, heterocycloalkyl, heteroaryl, arylcarbonyl, andheteroarylcarbonyl.R⁴ and R⁵ are, independently, H or C₁₋₆ alkyl, alkoxy, cycloalkyl,aryloxy, cycloalkoxy, aryl, heteroalkyl, heterocycloalkyl, heteroaryl,arylcarbonyl, and heteroarylcarbonyl or R⁴ and R⁵ together form acycloalkyl, cycloalkoxy, aryl, aryloxy, heterocycloalkyl, heteroaryl,arylcarbonyl, and heteroarylcarbonyl, any of which can be optionallysubstituted with alkyl, alkoxy, cycloalkyl, aryloxy, cycloalkoxy, aryl,heteroalkyl, heterocycloalkyl, heteroaryl, arylcarbonyl, andheteroarylcarbonyl;n is any integer from 0 to 6;or a pharmaceutically acceptable salt thereof.

In one embodiment, X and Y can be, independently, chlorine (Cl), bromine(Br) or iodine (I). In an exemplified embodiment, X is Cl and Y is Cl.

As used herein, alkyl means straight or branched chain, saturated ormono- or polyunsaturated hydrocarbon groups having from 1 to 20 carbonatoms and C_(1-X) alkyl means straight or branched chain alkyl groupscontaining from one up to X carbon atoms. For example, C₁₋₆ alkyl meansstraight or branched chain alkyl groups containing from one up to 6carbon atoms. Alkoxy means an alkyl-O-group in which the alkyl group isas previously described. Cycloalkyl includes a nonaromatic monocyclic ormulticyclic ring system, including fused and spiro rings, of from aboutthree to about 10 carbon atoms. A cyclic alkyl may optionally bepartially unsaturated. Cycloalkoxy means a cycloalkyl-O-group in whichcycloalkyl is as defined herein. Aryl means an aromatic monocyclic ormulticyclic carbocyclic ring system, including fused and spiro rings,containing from about six to about 14 carbon atoms. Aryloxy means anaryl-O-group in which the aryl group is as described herein.Alkylcarbonyl means a RC(O)— group where R is an alkyl group aspreviously described. Alkoxycarbonyl means an ROC(O)— group where R isan alkyl group as previously described. Cycloalkylcarbonyl means anRC(O)— group where R is a cycloalkyl group as previously described.Cycloalkoxycarbonyl means an ROC(O)— group where R is a cycloalkyl groupas previously described.

Heteroalkyl means a straight or branched-chain having from one to 20carbon atoms and one or more heteroatoms selected from nitrogen, oxygen,or sulphur, wherein the nitrogen and sulphur atoms may optionally beoxidized, i.e., in the form of an N-oxide or an S-oxide.Heterocycloalkyl means a monocyclic or multicyclic ring system (whichmay be saturated or partially unsaturated), including fused and spirorings, of about five to about 10 elements wherein one or more of theelements in the ring system is an element other than carbon and isselected from nitrogen, oxygen, silicon, or sulphur atoms. Heteroarylmeans a five to about a 14-membered aromatic monocyclic or multicyclichydrocarbon ring system, including fused and spiro rings, in which oneor more of the elements in the ring system is an element other thancarbon and is selected from nitrogen, oxygen, silicon, or sulphur andwherein an N atom may be in the form of an N-oxide. Arylcarbonyl meansan aryl-CO-group in which the aryl group is as described herein.Heteroarylcarbonyl means a heteroaryl-CO-group in which the heteroarylgroup is as described herein and heterocycloalkylcarbonyl means aheterocycloalkyl-CO-group in which the heterocycloalkyl group is asdescribed herein. Aryloxycarbonyl means an ROC(O)— group where R is anaryl group as previously described. Heteroaryloxycarbonyl means anROC(O)— group where R is a heteroaryl group as previously described.Heterocycloalkoxy means a heterocycloalkyl-O— group in which theheterocycloalkyl group is as previously described.Heterocycloalkoxycarbonyl means an ROC(O)— group where R is aheterocycloalkyl group as previously described.

Examples of saturated alkyl groups include, but are not limited to,methyl, ethyl, N-propyl, isopropyl, N-butyl, tert-butyl, isobutyl,sec-butyl, N-pentyl, N-hexyl, N-heptyl, and N-octyl. An unsaturatedalkyl group is one having one or more double or triple bonds.Unsaturated alkyl groups include, for example, ethenyl, propenyl,butenyl, hexenyl, vinyl, 2-propynyl, 2-isopentenyl, 2-butadienyl,ethynyl, 1-propynyl, 3-propynyl, and 3-butynyl. Cycloalkyl groupsinclude, for example, cyclopentyl, cyclohexyl, 1-cyclohexenyl,3-cyclohexenyl, and cycloheptyl. Heterocycloalkyl groups include, forexample, 1-piperidinyl, 2-piperidinyl, 3-piperidinyl, 3-morpholinyl,4-morpholinyl, tetrahydrofuran-2-yl, tetrahydrofuran-3-yl,tetrahydrothien-2-yl, tetrahydrothien-3-yl, 1-piperazinyl,2-piperazinyl, and 1,4-diazabicyclooctane. Aryl groups include, forexample, phenyl, indenyl, biphenyl, 1-naphthyl, 2-naphthyl, anthracenyl,and phenanthracenyl. Heteroaryl groups include, for example, 1-pyrrolyl,2-pyrrolyl, 3-pyrrolyl, furyl, thienyl, imidazolyl, oxazolyl, thiazolyl,pyrazolyl, pyridyl, indolyl, quinolinyl, isoquinolinyl, benzoquinolinyl,carbazolyl, and diazaphenanthrenyl.

As used herein, halogen means the elements fluorine (F), chlorine (Cl),Bromine (Br), and iodine (I).

The term pharmaceutically-acceptable salts means salts of the platinumcomplexes of the invention which are prepared with acids or bases,depending on the particular substituents present on the subjectcomplexes described herein. Examples of a pharmaceutically-acceptablebase addition salts include sodium, potassium, calcium, ammonium, ormagnesium salt. Examples of pharmaceutically-acceptable acid additionsalts include hydrochloric, hydrobromic, nitric, phosphoric, carbonic,sulphuric, and organic acids like acetic, propionic, benzoic, succinic,fumaric, mandelic, oxalic, citric, tartaric, maleic, and the like.Pharmaceutically-acceptable salts of platinum complexes of the inventioncan be prepared using conventional techniques.

It will be appreciated by those skilled in the art that certain of theplatinum complexes of the invention may contain one or moreasymmetrically substituted carbon atoms which can give rise tostereoisomers. All such stereoisomers, including enantiomers, anddiastereoisomers and mixtures, including racemic mixtures thereof, arecontemplated within the scope of the present invention.

Platinum complexes of the present invention are potent and selectivedisruptors of STAT activity. The complexes designated herein as CPA-7,CPA-10, CPA-39 (HK104), CPA-43 (HK106), CPA-46 (HK111), CPA-51 (HK110),CPA-55 (HK109), CPA-30 (HK112), and CPA-41 strongly disrupt Stat3activity and interfere with its ability to bind to its consensus bindingsequence. Platinum complexes of the invention can induce cell growthinhibition and apoptosis in transformed and tumor cells withpersistently active STATs. Malignant cells with aberrant or constitutiveSTAT signaling are highly sensitive to platinum complexes of theinvention. General cytotoxicity of the subject platinum complexes tonormal cells is minimal or nil. In addition, strong apoptosis is inducedby platinum compounds of the invention in malignant cells that harborpersistently-active STAT signaling, which correlates with suppression ofaberrant STAT activity in these cells.

Platinum complexes of the invention also exhibit anti-tumor activity inmelanoma and colon tumors in vivo. The abrogation ofconstitutively-active STATs in tumors treated with platinum complexes ofthe invention is consistent with their effects on STAT activity both invitro and in whole cells, and together establish STAT-based anti-tumoreffects of these compounds.

Methods of the invention comprise inhibiting function of a STAT bycontacting a cell expressing a STAT with a platinum complex of theinvention wherein the complex is taken in or otherwise provided insidethe cell. Platinum complexes of the invention can physically interactwith the DNA-binding domain of Stat3 and thereby disrupts its ability tobind to DNA. In Src-transformed mouse fibroblasts, as well as in humantumor cells of the breast, prostate, and mouse melanoma cells thatcontain constitutive Stat3 activity, both CPA-1 and CPA-7 abrogate Stat3signaling function and thereby induce cell growth inhibition andapoptosis.

Methods of the invention also comprise inhibiting the function and/orgrowth and replication of a cell that is aberrantly or constitutivelyexpressing a STAT, such as Stat1 or Stat3. In one embodiment, the methodcomprises contacting a cell with a platinum complex of the invention. Inone embodiment, the cell is a tumor cell, cancer cell, or a transformedcell. The cell can be a cell from a mammal, including human, monkey,chimpanzee, ape, dog, cat, cow, pig, and horse.

Platinum complexes of the invention can be delivered to a cell eitherthrough direct contact with the cell or via a carrier means. Carriermeans for delivering compositions to cells are known in the art andinclude, for example, encapsulating the composition in a liposomemoiety. Another means for delivery of platinum complexes of theinvention to a cell comprises attaching the platinum complexes to aprotein or nucleic acid that is targeted for delivery to the targetcell. Published U.S. Patent Application Nos. 20030032594 and 20020120100disclose amino acid sequences that can be coupled to another compositionand that allows the composition to be translocated across biologicalmembranes. Published U.S. Patent Application No. 20020035243 alsodescribes compositions for transporting biological moieties across cellmembranes for intracellular delivery.

The subject invention also concerns methods for treating oncological orinflammatory disorders in a patient. In one embodiment, an effectiveamount of a platinum complex of the present invention is administered toa patient having an oncological or inflammatory disorder and who is inneed of treatment thereof. Methods of the invention can optionallyinclude identifying a patient who is or may be in need of treatment ofan oncological or inflammatory disorder. The patient can be a human orother mammal, such as a primate (monkey, chimpanzee, ape, etc.), dog,cat, cow, pig, or horse, or other animals having an oncologicaldisorder. Means for administering and formulating platinum complexes foradministration to a patient are known in the art, examples of which aredescribed herein. Oncological disorders include cancer and/or tumors ofthe bone, breast, kidney, mouth, larynx, esophagus, stomach, testis,cervix, head, neck, colon, ovary, lung, bladder, skin, liver, muscle,pancreas, prostate, blood cells (including lymphocytes), and brain.Inflammatory disorders include arthritis, multiple sclerosis, lupus,Crohn's disease, and related neurological and inflammatory connectivetissue diseases (e.g., Sjögren's syndrome).

For the treatment of oncological disorders, the platinum complexes ofthis invention can be administered to a patient in need of treatment incombination with other antitumor or anticancer substances or withradiation therapy or with surgical treatment to remove a tumor. Theseother substances or radiation treatments may be given at the same as orat different times from the platinum complexes of this invention. Forexample, the platinum complexes of the present invention can be used incombination with mitotic inhibitors such as taxol or vinblastine,alkylating agents such as cyclophosamide or ifosfamide, antimetabolitessuch as 5-fluorouracil or hydroxyurea, DNA intercalators such asadriamycin or bleomycin, topoisomerase inhibitors such as etoposide orcamptothecin, antiangiogenic agents such as angiostatin, antiestrogenssuch as tamoxifen, and/or other anti-cancer drugs or antibodies, suchas, for example, GLEEVEC (Novartis Pharmaceuticals Corporation) andHERCEPTIN (Genentech, Inc.), respectively.

Many tumors and cancers have viral genome present in the tumor or cancercells. For example, Epstein-Barr Virus (EBV) is associated with a numberof mammalian malignancies. The platinum complexes of the subjectinvention can be used alone or in combination with anticancer orantiviral agents, such as ganciclovir, azidothymidine (AZT), lamivudine(3TC), etc., to treat patients infected with a virus that can causecellular transformation and/or to treat patients having a tumor orcancer that is associated with the presence of viral genome in thecells. The platinum complexes of the subject invention can also be usedin combination with viral based treatments of oncologic disease. Forexample, platinum complexes of the invention can be used with mutantherpes simplex virus in the treatment of non-small cell lung cancer(Toyoizumi et al., 1999).

The subject invention also concerns methods for treating bacterial andviral infections of a patient using a platinum complex of the invention.In one embodiment, an effective amount of a platinum complex of theinvention is administered to a patient having a bacterial or viralinfection. Methods of the invention can optionally include identifying apatient who is or may be in need of treatment of a bacterial or viralinfection. The patient can be a human or other mammal, such as a primate(monkey, chimpanzee, ape, etc.), dog, cat, cow, pig, or horse, or otheranimal infected with a bacteria or virus. Bacterial infections that canbe treated according to the present invention include those fromStaphylococcus, Streptococcus, Salmonella, Bacillus, Clostridium,Pseudomonas, Neisseria, Mycobacterium, and Yersinia. Viral infectionsthat can be treated according to the present invention include, but arenot limited to, those associated with human immunodeficiency virus(HIV), human T cell leukemia virus (HTLV), Papillomavirus (e.g., humanpapilloma virus), Polyomavirus (e.g., SV40, BK virus, DAR virus),orthopoxvirus (e.g., variola major virus (smallpox virus)), EBV, herpessimplex virus (HSV), hepatitis virus, Rhabdovirus (e.g., Ebola virus)and cytomegalovirus (CMV). Platinum compositions of the presentinvention can also be used to treat viral diseases in the presence ofphotodynamic therapy (Cuny et al., 1999). Platinum complexes of thepresent invention which can be used in photodynamic therapy include, butare not limited to, the complexes designated herein as CPA-30, CPA-32,CPA-38, CPA-39, CPA-41, CPA-42, CPA-43, CPA-45, CPA-46, CPA-51, CPA-53,CPA-54, CPA-55, and JP5. It is contemplated that these compounds areactivated by light to activate their antiviral, antibacterial,antitumor, antiparasitic, or cellular effects.

Platinum complexes of the subject invention can also be used to treatpatients infected with a parasitic organism. In one embodiment, thepatient is administered a therapeutically effective amount of a platinumcomplex of the present invention. Methods of the invention canoptionally include identifying a patient who is or may be in need oftreatment of a parasitic infection. The patient can be a human or othermammal, such as a primate (monkey, chimpanzee, ape, etc.), dog, cat,cow, pig, or horse, or other animal infected with a parasitic organism.Disease conditions that can be treated according to the presentinvention include, but are not limited to, leishmania, toxoplasmosis,schistosomiasis, trypanosomiasis, pneumocystis, malaria, andtrichinosis. Parasitic organisms that can cause disease conditionstreatable according to the present invention include, but are notlimited to, Leishmania, Toxoplasma, Schistosoma, Plasmodium, andTrypanosoma. The subject invention can also be used to treatgastrointestinal disorders caused by parasitic organisms such as,Entamoeba, Giardia, Trichomonas, and nematodes such as Ascaris,Trichuris, Enterobius, Necator, Ancylostoma, Strongyloides, andTrichinella. In another embodiment, a platinum complex of the presentinvention can be administered to patients prophylactically, wherein anuninfected patient is traveling to or will be present in an area whereparasitic disease is prevalent or poses a risk to the patient.Accordingly, the patient can be treated with a composition of thepresent invention prior to the patient's exposure to or presence in thearea where parasitic disease is prevalent or poses a risk and/or priorto infection with the parasitic organism.

Platinum complexes of the present invention can also be used to treatbiological products in vitro that are contaminated with or suspected ofbeing contaminated with a virus on a bacterial or parasitic organism.Biological products which can be treated with a platinum complexes ofthe present invention include, but are not limited to, whole blood,fractionated blood, plasma, serum, whole organs, or parts of organs, andcells, including blood cells, muscle cells, skin cells, and neuralcells, and products derived from cells. Products derived from cellswhich can be treated with a platinum complex of the present inventioninclude, but are not limited to, interferons, interleukins, bloodclotting factors such as factor VIII, IX, X, and the like, insulin,polyclonal and monoclonal antibodies, growth factors, cytokines, andother products. Treatment of biological products comprises contactingthe product for an effective amount of time and with an effective amountof a platinum complex of the present invention. If necessary, thebiological product can be subsequently washed, preferably with asuitable sterile wash solution such as phosphate buffered saline, toremove the platinum complex that was used to treat the product.

Therapeutic application of the subject platinum complexes, andcompositions containing them, can be accomplished by any suitabletherapeutic method and technique presently or prospectively known tothose skilled in the art. The subject platinum complexes can beadministered by any suitable route known in the art including, forexample, oral, nasal, rectal, and parenteral routes of administration.As used herein, the term parenteral includes subcutaneous, intradermal,intravenous, intramuscular, intraperitoneal, and intrasternaladministration, such as by injection. Administration of the subjectplatinum complexes of the invention can be continuous or at distinctintervals as can be readily determined by a person skilled in the art.

Platinum complexes of the subject invention can be formulated accordingto known methods for preparing pharmaceutically useful compositions.Formulations are described in detail in a number of sources which arewell known and readily available to those skilled in the art. Forexample, Remington's Pharmaceutical Science by E. W. Martin describesformulations which can be used in connection with the subject invention.In general, the compositions of the subject invention will be formulatedsuch that an effective amount of the bioactive platinum complex iscombined with a suitable carrier in order to facilitate effectiveadministration of the composition. The compositions used in the presentmethods can also be in a variety of forms. These include, for example,solid, semi-solid, and liquid dosage forms, such as tablets, pills,powders, liquid solutions or suspension, suppositories, injectable andinfusible solutions, and sprays. The preferred form depends on theintended mode of administration and therapeutic application. Thecompositions also preferably include conventional pharmaceuticallyacceptable carriers and diluents which are known to those skilled in theart. Examples of carriers or diluents for use with the subject platinumcomplexes include ethanol, dimethyl sulfoxide, glycerol, alumina,starch, and equivalent carriers and diluents. To provide for theadministration of such dosages for the desired therapeutic treatment,pharmaceutical compositions of the invention will advantageouslycomprise between about 0.1% and 99%, and especially, 1 and 15% by weightof the total of one or more of the subject platinum complexes based onthe weight of the total composition including carrier or diluent.

The platinum complexes of the subject invention can also be administeredutilizing liposome technology, slow release capsules, implantable pumps,and biodegradable containers. These delivery methods can,advantageously, provide a uniform dosage over an extended period oftime. The platinum complexes of the present invention can also beadministered in their salt derivative forms or crystalline forms knownto those of ordinary skill in the art.

The subject invention also concerns a packaged dosage formulationcomprising in one or more containers at least one platinum compound ofthe subject invention formulated in a pharmaceutically acceptabledosage.

All patents, patent applications, provisional applications, andpublications referred to or cited herein are incorporated by referencein their entirety, including all figures and tables, to the extent theyare not inconsistent with the explicit teachings of this specification.

Materials and Methods

Synthesis of NitroPlatinum (IV) Complexes.

Cis-diammineoplatinum(II) dichloride (cisplatin) can be purchased at99.9% purity from Sigma-Aldrich (#P4394). Using 0.300 grams of Cisplatin(0.00100 moles, FW=300.1), 150 mL of ultra deionized water and 50 mL ofdichloroethane are added to a 250-mL Erlenmeyer flask. However, hexaneor any organic solvents can be substituted in place of the dichlorethaneused here. The choice of a sixth ligand includes the availability of anitrogen, sulfur or oxygen atom in the chemical structure providing aLewis base for bonding to the oxidized Pt. Other bondings are possiblewith metals, halides (such as HCl) or through chelation or interactionwith pi molecular orbitals. One mole of the chosen ligand per mole ofcisplatin should be weighed and added to the mixture. Organic solvents,such as dichloroethane, provide solubility for organic ligands ofhydrophobic nature. A magnetic stir bar is placed in the mixture and theflask placed on a magnetic stir plate in a chemical fume hood. A lecturebottle of dinitrogen tetroxide is fitted with a regulator and Teflonhose, with a glass pipet attached to the hose outlet. The pipet tip isinserted into the lower solvent (e.g., dichloroethane) and the lecturebottle warmed slightly with a warm water bath. Nitrogen dioxide gas isreleased at a rate of approximately one bubble per second into thestirring mixture. The gas should be added until all the yellow cisplatinis consumed; the disappearance of yellow solids and yellow solution willindicate consumption of the available cisplatin. A blue color is notedto indicate formation of the nitrosyl intermediate; variations in hueand duration of this color have been observed. Gas addition is thenterminated (remove the pipet to prevent vacuum suction into the lecturebottle) and the flask covered in aluminum foil to prevent lightexposure. The flask should be left to stir overnight, uncovered.

Additional nitrogen dioxide may be added the next day to check forcompleteness of reaction. A blue color would indicate incompleteoxidation of platinum (II). Normally, this blue fades within tenminutes. For a colorless ligand, the solution has become yellowovernight. If blue color remains, allow it to continue stirring. Themixture requires air for complete oxidation, so should not be tightlycovered. Continued oxidation with air can be accelerated using air blownthrough a trap into the Erlenmeyer, over the liquids. The solvents willevaporate in about two days, leaving a yellow precipitate, which is theproduct.

The precipitate can be purified via recrystallization in methanol, DMSO,or other suitable solvent. Alternatively, the product can be purified onsilica columns or using HPLC.

MTT Assay.

1. Prepare a suspension of A549 cells at 2×10⁵ cells per mL insupplemented DMEM/F12 growth medium.

2. Plate 2×10⁴ cells per well in a 96 well cell culture plate by adding100 μL of stock suspension to each well.

3. For each platinum compound (already in solution), prepare a readilyusable stock solution in DMEM/F12 medium.

4. For each compound generate triplicate trials of 0, 10, 20, 30, 40,50, 60, and 70 μM concentration. This is achieved in situ by addingappropriate volumes of stock solution to each well along with the volumeof untreated medium necessary to generate the desired concentration in afinal volume of 200 μL.

5. Gently agitate plates to mix contents. Incubate at 37° C., 7% CO₂ for45 hours.

6. Add 20 μL of 5 mg/mL MTT solution (in PBS) to each well.

7. Gently agitate plates to mix contents and incubate an additional 3hours to allow product development.

8. Remove plates from incubator and agitate to cause settling offormazan product.

9. Aspirate out liquid contents of each well using needle and syringeand discard.

10. Add 200 μL DMSO to each well to dissolve formazan product.

11. Agitate plates until all of the formazan product is in solution andno purple crystals remain on the bottom surface of the wells.

12. Read the absorbance of each well at 475 mm using Varian software forCary 50 UV-vis Spectrophotometer with fiber optic probe accessory.

XTT Assay.

A 96-well plate was used for the assays. Approximately 2.5×10⁴ cells inlog phase were added to each well. A platinum complex of the inventionwas dispensed into each well (dissolved in 20% DMSO and 80% media), withadditional media added as needed to account for uniform volumes. Controlwells contained only cells and media. Each concentration assay wasperformed in triplicate. Plates were incubated for 48 hours at 37° C.with 7.5% CO₂. XTT from MD Biosciences. Quebec, was then added accordingto the provided protocol concentrations and allowed to react for 3hours. Plates were agitated 5 minutes before reading absorbance at 475nm on a Varian Cary 50 spectrophotometer with a fibre-optic probe.Percent survival as compared to control wells was plotted againstplatinum complex concentration.

Nuclear Extract Preparation and Analysis by EMSA of HK-DesignatedPlatinum Complexes.

Nuclear extracts were prepared from NIH3T3/hEGFR cell that overexpresshuman epidermal growth factor (EGF) receptor and stimulated with EGF (6ng/ml) for 15 min. Nuclear extracts were pre-incubated with compoundsfor 30 min at room temperature prior to incubation with radiolabeledprobe. The ³²P-labeled oligonucleotide probe used is hSIE (high affinitysis-inducible element, m67 variant, 5′-AGCTTCATTTCCCGTAAATCCCTA-3′) (SEQID NO:1) that binds both Stat1 and Stat3.

Following are examples which illustrate procedures for practicing theinvention. These examples should not be construed as limiting. Allpercentages are by weight and all solvent mixture proportions are byvolume unless otherwise noted.

Example 1 MTT Assay Data for Platinum Complexes

MTT assays are used for the quantitative determination of cellularproliferation and activation and for the quantitation of in vitro tumorcell sensitivity to anti-cancer compounds. The assay is based on thecleavage of the yellow tetrazolium salt MTT into purple colored formazanby metabolically active cells. Solubilized formazan product can bephotometrically quantitated using an ELISA plate reader. A decrease inthe number of living cells results in a decrease in total metabolicactivity which leads to a decrease in color formation. Platinumcomplexes of the present invention were tested in MTT assays using A549cells to determine anti-cancer cell activity. The results are shown inTables 1, 2, 3, and 4 and FIGS. 1, 2 and 4. Table 1 shows percentsurvival of A549 cells and Table 2 shows the IC50 from the data inTable 1. FIGS. 1, 2, and 4 show percent survival versus platinum complexconcentration in graphical form. FIG. 6 shows a graph of absorbance at570 nm versus concentration of several platinum complexes (μm) of theinvention.

TABLE 1 Percent of Survival of A549 Using Platinum Complex in MTT AssayConcentration Percent Average Complex (in μM) Survival Absorbance Cis-Pt10 98.2423 2.3196 20 98.3779 2.3228 30 99.6400 2.3526 40 90.2757 2.131550 78.1288 1.8447 60 46.7536 1.1039 70 30.1470 0.7118 CPA-7 10 99.10212.3399 20 100.0974 2.3634 30 90.6908 2.1413 40 46.9993 1.1097 50 20.07120.4739 60 28.2750 0.6676 70 24.3742 0.5755 CPA-46 (Rhodamine) 10101.7831 2.4032 20 97.3190 2.2978 30 96.0188 2.2671 40 78.8531 1.8618 5043.5602 1.0285 60 29.8208 0.7041 CPA-31 (Citrate) 10 102.8080 2.4274 2099.5087 2.3495 30 100.7073 2.3778 40 50 not performed 60 70 CPA-43(Dinitrophenyl 10 102.2828 2.4150 Hydrazine) 20 78.5820 1.8554 3090.8517 2.1451 40 55.1861 1.3030 50 60 not performed 70 CPA-41 (Diphenyl10 100.4024 2.3706 Isobenzofuran) 20 100.9148 2.3827 30 89.4668 2.112440 36.6270 0.8648 50 60 not performed 70 CPA-55 (Hydroorotic Acid) 10103.6847 2.4481 20 100.5082 2.3731 30 98.5473 2.3268 40 58.3626 1.378050 60 not performed 70 CPA-39 (Fluorescein) 10 107.2085 2.5313 20102.9478 2.4307 30 99.9492 2.3599 40 83.0587 1.9611 50 43.7000 1.0318 6032.9338 0.7776 70 41.4976 0.9798 CPA-51 (Luminol) 10 105.5652 2.4925 20109.1017 2.5760 30 98.3186 2.3214 40 87.3152 2.0616 50 58.7396 1.3869 6037.3004 0.8807 70 45.8219 1.0819

TABLE 2 Calculated IC50's from MTT Assay Data of Table 1 Calculated IC50 Complex (uM concentration) Cisplatin 63.29 CPA-51 (Luminol) 61.21CPA-39 (Fluorescein) 56.39 CPA-46 (Rhodamine) 52.88 CPA-55 (HydrooroticAcid) 54.20 Extrapolated from data 10-40 uM CPA-43 (DNP) 49.59Extrapolated from data 10-40 uM CPA-41 (IBF) 40.71 Extrapolated fromdata 10-40 uM CPA-7 45.43 CPA-31 (Citrate) 505.81 Suspected error due toincomplete data set Values in bold are outside the measured range. Allvalues are determined using a linear trend from percent survival vs.concentration data.

TABLE 3 Percent of Survival of A549 Using Platinum Complex in MTT AssayConcentration Percent Average Complex (in μM) Survival Absorbance Cis-Pt10 100.9661 2.4665 20 100.5199 2.4556 30 92.8650 2.2686 40 71.94321.7575 50 52.5114 1.2828 60 32.6293 0.7971 70 27.5001 0.6718 CPA-7 10100.1965 2.4477 20 95.3703 2.3298 30 57.9148 1.4148 40 18.7646 0.4584 5017.6880 0.4321 60 20.5166 0.5012 70 14.2454 0.3480 CPA-46 (Rhodamine) 10105.5017 2.5773 20 98.7269 2.4118 30 80.3840 1.9637 40 49.3021 1.2044 5025.7153 0.6282 60 26.3867 0.6446 70 25.2364 0.6165 CPA-31 (Citrate) 10103.5409 2.5294 20 97.7322 2.3875 30 73.5929 1.7978 40 33.5503 0.8196 5024.7616 0.6049 60 24.8352 0.6067 70 25.4943 0.6228 CPA-43 (Dinitrophenyl10 105.7759 2.5840 Hydrazine) 20 93.8311 2.2922 30 60.7188 1.4833 4026.5218 0.6479 50 18.9979 0.4641 60 26.6241 0.6504 70 20.7090 0.5059CPA-41 (Diphenyl 10 100.2906 2.4500 Isobenzofuran) 20 97.4457 2.3805 3060.1171 1.4686 40 17.2336 0.4210 50 14.9290 0.3647 60 22.7148 0.5549 7022.2891 0.5445 CPA-55 (Hydroorotic 10 100.6468 2.4587 Acid) 20 101.82572.4875 30 69.1883 1.6902 40 26.6200 0.6503 50 20.0336 0.4894 60 28.99010.7082 70 23.4680 0.5733 CPA-39 (Fluorescein) 10 108.7069 2.6556 20100.7655 2.4616 30 85.5295 2.0894 40 55.5405 1.3568 50 26.8779 0.6566 6024.9130 0.6086 70 25.2405 0.6166 CPA-51 (Luminol) 10 101.2895 2.4744 20101.7970 2.4868 30 85.9552 2.0998 40 60.4118 1.4758 50 38.2824 0.9352 6028.9983 0.7084 70 28.8919 0.7058

TABLE 4 Percent of Survival of A549 Using Platinum Complex in MTT AssayConcentration Percent Average Complex (in μM) Survival Absorbance Cis-Pt10 83.09927847 0.5299 20 103.1095973 0.6575 30 77.94510544 0.49703333340 74.69369001 0.4763 50 57.14022853 0.364366667 60 44.12933942 0.281470 29.06932676 0.185366667 CPA-7 10 20 91.22258971 0.5817 30 93.977406320.599266667 40 45.5459529 0.290433333 50 17.51164263 0.111666667 603.538920018 0.022566667 70 3.538920018 0.022566667 CPA-44 (Succinamide)10 54.96042107 0.350466667 20 23.37150871 0.149033333 30 5.4939511650.035033333 40 4.929396716 0.031433333 50 6.424420535 0.040966667 604.8718958 0.031066667 70 5.164627736 0.032933333

TABLE 5

Example 2 XTT Assay Data for Platinum Complexes

The XTT assay is based on the conversion of the yellow tetrazalium saltXTT into an orange formazan dye by metabolically active cells. Theorange formazan dye is soluble and can be photometrically quantitatedusing an ELISA plate reader. A decrease in the number of living cellsresults in a decrease in total metabolic activity which leads to adecrease in color formation. Platinum complexes of the present inventionwere tested in XTT assays using A549 cells to determine anti-cancer cellactivity. The percent survival of cells versus platinum complexconcentration is shown in graphical form in FIGS. 3 and 7.

Example 3 Reduction of CPA-7 Platinum Complex by Glutathione

FIGS. 5A-C show the inertness of CPA-7 to glutathione reduction. In thefigures, 20 μm CPA-7 was added in PBS/20% DMSO with 10 mM glutathione.The readings were initiated in late evening where data points werecollected every 10 minutes at the lambda max for CPA-7, 226 nm. The datashows slow reduction which then virtually stops during the night hours,and resumes the next day. This data demonstrates sensitivity to lightand stability against GSH reduction. Data was collected on a Varian Cary50 fitted with a fibreoptic probe in the kinetics mode. The Cary 50 usesa pulsed Xenon lamp with discontinuous irradiation between readings. A25-mL volumetric flask was used for the solution with a magneticstirring bar at slow speed.

Example 4 MTT Assay Data for Platinum Complexes

MTT assays are used for the quantitative determination of cellularproliferation and activation and for the quantitation of in vitro tumorcell sensitivity to anti-cancer compounds. The assay is based on thecleavage of the yellow tetrazolium salt MTT into purple colored formazanby metabolically active cells. Solubilized formazan product can bephotometrically quantitated using an ELISA plate reader. A decrease inthe number of living cells results in a decrease in total metabolicactivity which leads to a decrease in color formation. Platinumcomplexes of the present invention were tested in MTT assays using A549cells to determine anti-cancer cell activity. The results are shown inTables 6, 7, 8, and 9 and FIGS. 8, 9, and 11. Table 6 shows percentsurvival of A549 cells and Table 7 shows the IC50 from the data in Table6. FIGS. 8, 9, and 11 show percent survival versus platinum complexconcentration in graphical form.

TABLE 6 MTT Assay: Percent of Survival of A549 Using cisplatin (Cis-Pt)or a Platinum Complex of formula:

Complex Concentration (in μM) Percent Survival Average Absorbance Cis-Pt10 98.2423 2.3196 20 98.3779 2.3228 30 99.6400 2.3526 40 90.2757 2.131550 78.1288 1.8447 60 46.7536 1.1039 70 30.1470 0.7118 A = Safranin 1* 1098.9412 2.3361 20 100.1440 2.3645 30 101.8000 2.4036 40 85.0705 2.008650 82.5674 1.9495 60 69.5989 1.6433 70 45.1484 1.0660 A = Safranin 2* 10101.2791 2.3913 20 101.2409 2.3904 30 95.5826 2.2568 40 83.2027 1.964550 81.0131 1.9128 60 67.3203 1.5895 70 39.1682 0.9248 A = Methylene blue10 100.5887 2.3750 20 97.1030 2.2927 30 95.3369 2.2510 40 66.5029 1.570250 36.1781 0.8542 60 50.4468 1.1911 70 40.2567 0.9505 A =6-Aminonicotinamide 10 100.5548 2.3742 20 103.0071 2.4321 30 96.62452.2814 40 72.9025 1.7213 50 36.4999 0.8618 60 32.7771 0.7739 70 36.15260.8536 A = Methyl alpha-D- 10 104.1125 2.4582 Mannopyranoside 20104.2311 2.4610 30 91.2456 2.1544 40 46.6096 1.1005 50 28.0844 0.6631 6039.8967 0.9420 70 35.9112 0.8479 *Two different isolates of the platinum(IV) complex substituted with Safranin were isolated (referred to hereinas Safranin 1 and Safranin 2) and tested in the assay.

TABLE 7 Calculated IC50's from MTT Assay Data of Table 1 Calculated IC50 Complex (uM concentration) Safranin 1* 78.60 Safranin 2* 72.57Methylene blue 56.36 6-Aminonicotinamide 53.06 Methyl alpha-D- 50.10Mannopyranoside Values in bold are outside the measured range. Allvalues are determined using a linear trend from percent survival vs.concentration data. *Two different isolates of the platinum (IV) complexsubstituted with Safranin were isolated (referred to herein as Safranin1 and Safranin 2) and tested in the assay.

TABLE 8 MTT Assay: Percent of Survival of A549 Using cisplatin (Cis-Pt)or a Platinum Complex of formula:

Complex Concentration (in μM) Percent Survival Average Absorbance Cis-Pt10 100.9661 2.4665 20 100.5199 2.4556 30 92.8650 2.2686 40 71.94321.7575 50 52.5114 1.2828 60 32.6293 0.7971 70 27.5001 0.6718 A =Safranin 1* 10 100.3152 2.4506 20 101.0725 2.4691 30 100.9661 2.4665 4085.6236 2.0917 50 62.0533 1.5159 60 47.8079 1.1679 70 35.5807 0.8692 A =Safranin 2* 10 101.3304 2.4754 20 101.0766 2.4692 30 94.1790 2.3007 4083.7284 2.0454 50 62.7001 1.5317 60 46.0928 1.1260 70 35.4456 0.8659 A =Methylene blue 10 103.5613 2.5299 20 88.6365 2.1653 30 67.6900 1.6536 4030.7913 0.7522 50 25.7972 0.6302 60 31.5240 0.7701 70 30.6234 0.7481 A =6- 10 101.1544 2.4711 Aminonicotinamide 20 100.9947 2.4672 30 81.44421.9896 40 44.8770 1.0963 50 26.5463 0.6485 60 23.7873 0.5811 70 25.76450.6294 A = Methyl alpha-D 10 106.9098 2.6117 Mannopyranoside 20 99.68892.4353 30 70.8789 1.7315 40 25.2691 0.6173 50 17.8886 0.4370 60 20.53710.5017 70 21.8552 0.5339 *Two different isolates of the platinum (IV)complex substituted with Safranin were isolated (referred to herein asSafranin 1 and Safranin 2) and tested in the assay.

TABLE 9 MTT Assay: Percent of Survival of A549 Using cisplatin (Cis-Pt)or a Platinum Complex of formula:

Complex Concentration (in μM) Percent Survival Average Absorbance Cis-Pt10 83.09928 0.5299 20 103.1096 0.6575 30 77.94511 0.497033 40 74.693690.4763 50 57.14023 0.364367 60 44.12934 0.2814 70 29.06933 0.185367 A =Caffeine 10 72.23683268 0.460633333 20 61.71416504 0.393533333 3031.61504914 0.2016 40 13.94658583 0.088933333 50 7.814897233 0.04983333360 9.571288852 0.061033333 70 8.003082049 0.051033333 A = Safranin 1077.35441421 0.493266667 20 68.90177955 0.439366667 30 34.458730810.219733333 40 11.68836804 0.074533333 50 8.301041342 0.052933333 606.774653387 0.0432 70 8.541499718 0.054466667 A = Theophylline 1089.09505581 0.568133333 20 60.59551085 0.3864 30 33.86281222 0.21593333340 9.174009795 0.0585 50 0.737057197 0.0047 60 4.97644292 0.031733333 70−0.245685732 −0.001566667 A = N-acetyl glucosamine 10 96.894270970.617866667 20 59.27298978 0.377966667 30 27.21361538 0.173533333 403.75846897 0.023966667 50 6.225781006 0.0397 60 8.661728906 0.05523333370 2.974365569 0.018966667

Example 5 XTT Assay Data for Platinum Complexes

The XTT assay is based on the conversion of the yellow tetrazalium saltXTT into an orange formazan dye by metabolically active cells. Theorange formazan dye is soluble and can be photometrically quantitatedusing an ELISA plate reader. A decrease in the number of living cellsresults in a decrease in total metabolic activity which leads to adecrease in color formation. Platinum complexes of the present inventionwere tested in XTT assays using A549 cells to determine anti-cancer cellactivity. The percent survival of cells versus platinum complexconcentration is shown in graphical form in FIG. 10.

Example 6 Inhibition of In Vitro Stat3 DNA-Binding Activity byHK-Designated Platinum complexes

Other platinum (IV) complexes were evaluated for inhibitory activityagainst STAT DNA-binding in vitro. Analysis by EMSA of nuclear extractsprepared from EGF-stimulated fibroblast that activates Stat1, Stat3 andStat5 shows that preincubation (of extracts of equal total protein) withdifferent concentrations of platinum complexes for 30 min prior toincubation with ³²P-labeled hSIE probe results in a dose-dependentreduction in the levels of DNA-binding activities of Stat3 and Stat1(FIGS. 12A and 12B).

It should be understood that the examples and embodiments describedherein are for illustrative purposes only and that various modificationsor changes in light thereof will be suggested to persons skilled in theart and are to be included within the spirit and purview of thisapplication and the scope of the appended claims.

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1. A method for synthesis of a platinum complex, said method comprising:a) mixing cisplatin in water and an organic solvent; b) mixing into themixture of step (a) a ligand capable of bonding to the platinum ofcisplatin to form the platinum complex product; c) contacting themixture of step (b) with nitrogen dioxide gas; and d) separating theplatinum complex product from the solvent.
 2. The method according toclaim 1, wherein the organic solvent is dichloroethane or hexane.
 3. Themethod according to claim 1, wherein the platinum complex product isseparated from the solvent by evaporation of the solvent.
 4. The methodaccording to claim 1, wherein following step (d), the platinum complexproduct is further purified.
 5. The method according to claim 4, whereinthe platinum complex product is further purified by recrystallization ina solvent.
 6. The method according to claim 4, wherein the platinumcomplex product is further purified by adsorption on a silica column orby high performance liquid chromatography (HPLC).
 7. The methodaccording to claim 1, wherein the platinum complex has the structureshown in formula I:

wherein X and Y are, independently, any halogen, —NO₂, —ONO, or thestructure:

or X and Y together form the structure:

R¹ is —NO₂ or —ONO; R² is any halogen, —OH, —ONO, —ONO₂, —COR¹⁰,—OPO₃R¹⁰R¹¹, —OSO₃H, —OSeOOH, —SeOOH, —AsO₂, —OAsO₂, —NR¹⁰R¹¹,—NHR¹⁰R¹¹, —OOCR¹⁵, alkyl, alkoxy, cycloalkyl, cycloalkoxy, aryl,aryloxy, alkycarbonyl, alkoxycarbonyl, cycloalkylcarbonyl, heteroalkyl,heterocycloalkyl, heterocycloalkylcarbonyl, heteroaryl, arylcarbonyl,heteroarylcarbonyl, aryloxycarbonyl, heteroaryloxycarbonyl,heterocycloalkoxy, or heterocycloalkoxycarbonyl, any of which can beoptionally substituted with any halogen, —COOH, —OH, —NO₂, —NH₂,—N-alkyl, alkyl, alkoxy, cycloalkyl, cycloalkoxy, aryl, aryloxy,alkycarbonyl, alkoxycarbonyl, cycloalkylcarbonyl, heteroalkyl,heterocycloalkyl, heterocycloalkylcarbonyl, heteroaryl, arylcarbonyl,heteroarylcarbonyl, aryloxycarbonyl, heteroaryloxycarbonyl,heterocycloalkoxy, or heterocycloalkoxycarbonyl, or the structure:

any of which can be substituted with any halogen, —NH₂, —COOH, —OH,alkoxy, cycloalkoxy; R³ is, independently, —NH₃, or —NHR⁷; R⁷ is H, C₁₋₆alkyl, alkoxy, or aryl, optionally substituted with —NO₂ or —COOH; R¹⁰and R¹¹ are, independently, H, —NH₂, —OH, —NHR⁷, CONHR⁷, CON(R⁷), C₁₋₆alkyl, aryl, or heteroaryl, any of which can be optionally substitutedwith any halogen, —COOH, —OH, —NO₂, —NH₂, alkyl, alkoxy, cycloalkyl,cycloalkoxy, aryl, aryloxy, alkylcarbonyl, alkoxycarbonyl,cycloalkylcarbonyl, heteroalkyl, heterocycloalkyl,heterocycloalkylcarbonyl, heteroaryl, arylcarbonyl, heteroarylcarbonyl,aryloxycarbonyl, heteroaryloxycarbonyl, heterocycloalkoxy, orheterocycloalkoxycarbonyl; R¹⁵ is alkyl, alkoxy, cycloalkyl,cycloalkoxy, aryl, aryloxy, alkylcarbonyl, alkoxycarbonyl,cycloalkylcarbonyl, heteroalkyl, heterocycloalkyl,heterocycloalkylcarbonyl, heteroaryl, arylcarbonyl, heteroarylcarbonyl,aryloxycarbonyl, heteroaryloxycarbonyl, heterocycloalkoxy, orheterocycloalkoxycarbonyl, any of which can be optionally substitutedwith any halogen, —COOH, —OH, —NO₂, —NH₂, —N-alkyl, alkyl, alkoxy,cycloalkyl, cycloalkoxy, aryl, aryloxy, alkylcarbonyl, alkoxycarbonyl,cycloalkylcarbonyl, heteroalkyl, heterocycloalkyl,heterocycloalkylcarbonyl, heteroaryl, arylcarbonyl, heteroarylcarbonyl,aryloxycarbonyl, heteroaryloxycarbonyl, heterocycloalkoxy, orheterocycloalkoxycarbonyl; or a pharmaceutically acceptable saltthereof.
 8. The method according to claim 7, wherein X and Y are,independently, selected from the group consisting of F, Cl, Br, and I.9. The method according to claim 7, wherein X and Y are both Cl.
 10. Themethod according to claim 7, wherein R¹ is —NO₂.
 11. The methodaccording to claim 7, wherein R³ is —NH₃.
 12. The method according toclaim 1, wherein the platinum complex has the structure shown in formulaIII or formula IV:

wherein X and Y are, independently, any halogen, —NO₂, —ONO, or X and Ytogether form the structure:

R⁶ is, independently, NO₂, N, NH, or NH₂; R⁸ and R⁹ are, independently,H, C₁₋₆ alkyl, or —OH, any of which can be optionally substituted withany halogen, —COOH, —OH, —NO₂, —NH₂, alkyl, alkoxy, cycloalkyl,cycloalkoxy, aryl, aryloxy, alkycarbonyl, alkoxycarbonyl,cycloalkylcarbonyl, heteroalkyl, heterocycloalkyl,heterocycloalkylcarbonyl, heteroaryl, arylcarbonyl, heteroarylcarbonyl,aryloxycarbonyl, heteroaryloxycarbonyl, heterocycloalkoxy, orheterocycloalkoxycarbonyl; R¹² and R¹³ are, independently, H or C₁₋₆alkyl, or R¹² and R¹³ together form an aryl, cycloalkyl,heterocycloalkyl, or heteroaryl, any of which can be optionallysubstituted with any halogen, —COOH, —OH, —NO₂, —NH₂, alkyl, alkoxy,cycloalkyl, cycloalkoxy, aryl, aryloxy, alkycarbonyl, alkoxycarbonyl,cycloalkylcarbonyl, heteroalkyl, heterocycloalkyl,heterocycloalkylcarbonyl, heteroaryl, arylcarbonyl, heteroarylcarbonyl,aryloxycarbonyl, heteroaryloxycarbonyl, heterocycloalkoxy, orheterocycloalkoxycarbonyl; n is any integer from 0 to 6; or apharmaceutically acceptable salt thereof.
 13. The method according toclaim 1, wherein the platinum complex has the structure shown in formulaV or formula VI:

wherein X and Y are, independently, any halogen, —OH, H₂O, or —SO(CH₃)₂;and A can be any of the following:

and wherein R¹ is, independently, NH₂ or NH; R² and R³ are,independently, H, —OH, C₁₋₆ alkyl, alkoxy, cycloalkyl, aryloxy,cycloalkoxy, aryl, heteroalkyl, heterocycloalkyl, heteroaryl,arylcarbonyl, and heteroarylcarbonyl, any of which can be optionallysubstituted with alkyl, alkoxy, cycloalkyl, aryloxy, cycloalkoxy, aryl,heteroalkyl, heterocycloalkyl, heteroaryl, arylcarbonyl, andheteroarylcarbonyl; R⁴ and R⁵ are, independently, H or C₁₋₆ alkyl,alkoxy, cycloalkyl, aryloxy, cycloalkoxy, aryl, heteroalkyl,heterocycloalkyl, heteroaryl, arylcarbonyl, and heteroarylcarbonyl or R⁴and R⁵ together form a cycloalkyl, cycloalkoxy, aryl, aryloxy,heterocycloalkyl, heteroaryl, arylcarbonyl, and heteroarylcarbonyl, anyof which can be optionally substituted with alkyl, alkoxy, cycloalkyl,aryloxy, cycloalkoxy, aryl, heteroalkyl, heterocycloalkyl, heteroaryl,arylcarbonyl, and heteroarylcarbonyl; n is any integer from 0 to 6; or apharmaceutically acceptable salt thereof.
 14. The method according toclaim 13, wherein X and Y are, independently, selected from the groupconsisting of F, Cl, Br, and I.
 15. The method according to claim 13,wherein X and Y are both Cl.
 16. The method according to claim 13,wherein the platinum complex is selected from the group consisting of


17. The method according to claim 1, wherein the platinum complex hasthe structure: